Abstracts from the 17 th Annual Meeting of the German Society of Cytometry (DGfZ)
2007; Wiley; Volume: 71A; Issue: 9 Linguagem: Inglês
10.1002/cyto.a.20451
ISSN1552-4930
Autores Tópico(s)Cell Image Analysis Techniques
ResumoAbstracts from the 17th Annual Meeting of the German Society of Cytometry (DGfZ) 10–13 October 2007 Hosted at the University Hospital Regensburg, Germany Extraordinary Sponsorship by Regensburger Universitäatsstiftung Hans Vielberth Partec GmbH, Müunster, Germany In the capacity as president of the German Society of Cytometry, I would like to warmly welcome you to our 17th annual meeting. This year the society's meeting is being held for the very first time in Regensburg and is hosted by the University of Regensburg Medical Center. After 14 meetings traditionally held at the German Cancer Research Center (DKFZ) in Heidelberg, we moved the conference site to Leipzig where we had two successful meetings hosted at the Helmholtz Center for Environmental Research (UFZ). Since then our society has undergone a modification in both content and form. Over the last couple of years new ideas have influenced and reshaped the society and this modified body of thought has been reflected in the scientific programs presented in 2005 and 2006. Now in 2007, we have moved to Regensburg and intend to continue this trend of successful events by presenting a stimulating and rewarding scientific meeting held in charming, historic Regensburg. This year the annual meeting takes place for the very first time in Regensburg (Bavaria, Germany) and is hosted by the University of Regensburg Medical Center. I am convinced that our society has found a very appropriate and likewise extraordinarily attractive venue for the 17th annual meeting. The natural scientific landscape comprises the communities of the University, the Technical College and the University Medical Center with its associated Tumor Center, as well as the Archaea Center, the Technical School for Boundary Layer Chemistry and the WHO Collaborating Center, all situated on one common campus with a park-like character. The Technology Transfer Sites, the Data Processing Center and BioPark Regensburg, completed in the year 2000, are all within walking distance. The University Medical Center itself is constantly being enlarged and complemented by additional departments and modern research facilities. In 2006 Regensburg was selected by UNESCO as a World Cultural Heritage Site. Located on the Danube River, the medieval town contains many buildings of exceptional quality that testify to its history as an important trading center and to its influence in the region dating back to as early as the 9th century. It has a notable number of preserved historic structures spanning some two millennia, including ancient Roman, Romanesque and Gothic buildings. Regensburg's 11th–13th century architecture, including the market, City Hall and St. Peter's Cathedral, still defines the character of the town marked by tall towers, charming narrow lanes, and strong fortifications. The buildings include medieval Patrician houses and towers, a large number of churches and monastaries as well as the famous Stone Bridge, which dates back from the 12th century. The town is also remarkable for the vestiges that testify to its rich institutional and religious history as one of the centers of the Holy Roman Empire. However, Regensburg is anything but a museum! It is a lovable, livable and vital town in the center of Bavaria. Our society was originally founded by Cess Cornelisse, Georg Feichter, Wolfgang Goehde, Klaus Goerttler, Holger Hoehn, Andreas Radbruch, Peter Schwarzmann, and Günter Valet in 1989 and designated as the Society of Cytometry (Gesellschaft fuer Zytometrie, GZ). An association was born dedicated to providing an interdisciplinary platform for scientists principally interested in the field of flow and image cytometry. Founding members were scientists whose personal scientific development was, and still is, closely intertwined with the development and advancement of cytometric technologies in Europe. Since its foundation, annual meetings have been organized to provide a platform for interdisciplinary exchange in basic research, as well as clinical and industrial developments. In 1994 the original name of the society was changed to the Deutsche Gesellschaft fuer Zytometrie (DGfZ, German Society of Cytometry), however, over time the society has expanded to include increasing international participation and in the mid-nineties the conference language was changed to English. Since its foundation, the DGfZ has offered an organized platform and structure for the growing science of Cytometry. The focus of the scientific interest of DGfZ activities is the analysis of genetic, physiological and structural processes in cells. Ab initio, the DGfZ has aimed to promote methodological and technical innovations in flow cytometry (FCM), image cytometry (ICM) and slide based cytometry (SBC), which furthers understanding of the cell and its integration into multi-cellular systems. The spectrum ranges from Oncology, Immunology and Pathology, to Cytogenetics, Microbiology and Plant Culture as well as Ecology, and includes all areas where biological cells and tissues are the primary focus. The DGfZ is dedicated to providing a platform for interdisciplinary and scientific exchange, and to facilitate communication as well as the sharing of knowledge. The society has continuously engaged in innovative science and the development of cutting-edge technologies. Examples are the first commercially available flow cytometer as well as methods for dual laser flow cytometry, cell separation and sorting, among others. Even if the name “German Society of Cytometry” might suggest a technically oriented community, the society is consistently evolving. After extensive discussions in the mid-nineties, the council board decided to retain the original name in order to give shape to a society with a historical origin. Keeping pace with the times, however, a new way of thinking was reflected by the scientific topics the council board compiled and - needless to say - by the scientific presentations contributed by the conference attendees. Stem cell research, chip and array technology, and systems biology all became a vital part of past meetings representing cutting-edge research over a wide range of scientific fields. As a guiding force, the DGfZ provides a unique platform for a variety of scientists working in different areas of research and having different scientific interests, but all sharing the utilization of cytometric-based technologies and approaches to address scientific questions of critical importance. Günter Valet (previously affiliated to the Max Planck Institute, Martinsried, Germany, Dept. of Cell Biochemistry), officiated as the second president of the society from 1992 thru 1994 and has been deeply involved in the DGfZ since its foundation and is one of the pioneers who aptly realized that cytometric analysis had the potential for utilization in higher levels of research. He led cytometrists to the idea of cytomics by suggesting the application of the multimolecular analysis of cells and the heterogeneity of cell systems in combination with exhaustive bioinformatic knowledge extraction. Cytomics links proteomics and genomics to cell and tissue function by integrating the morphology and architecture of cell systems. In clinical applications, it opened the way towards predictive and preventive medicine in terms of individualized medicine. The idea of cytomics can be realized today by taking advantage of highly sophisticated cytometric technologies and, insofar, this innovative way of thinking has significantly contributed to a modified self-concept of the DGfZ. We very much appreciate the valuable and vital input given by Günter Valet to the DGfZ over many years. He has consistently offered his expertise in helping to reshape and reform the society, helping it to achieve its current status. Hence we would like to appoint Günter Valet this year as an honorary member of our society. Cancer Biology and Therapy in vivo and in situ Imaging Stem Cell Biology Analysis of Tissues and Tissue Related Systems Clinical Cytometry and Advances in Diagnostic Immunophenotyping Cytometry in Microbiology, Biotechnology, and Plants Cytometry in Systems Biology Novel Instrumentation and Applications Biosensoric Applications and Nanotechnologies The last topic listed has been integrated as a first-off topic this year from which we can expect innovative input for our meeting. Robert F. Murphy, Biological Sciences, Biomedical Engineering, and Machine Learning Director, Center for Bioimage Informatics Director, Joint CMU-Pitt Ph.D. Program in Computational Biology President-elect, International Society for Analytical Cytology Carnegie Mellon University, Pittsburgh, PA 15213, USA Thomas M. Jovin, Dept. of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany Leoni Kunz-Schughart, Technical University of Dresden, Faculty of Medicine, OncoRay, Dresden, Germany Thomas Ried, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Mario Roederer, Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, MD, USA Herbert Stepp, Laser Research Center, University Hospital Großhadern, LMU Munich, Germany János Szöllõsi, Medical and Health Science Center, Dept. of Biophysics and Cell Biology, University of Debrecen, Hungary Dieter Weiss, Dept. for Animal Physiology, University of Rostock, Germany Johannes Wessels, Center for Internal Medicine, Dept. of Nephrology and Rheumatology, University Hospital, Göttingen, Germany Otto Wolfbeis, Institute of Analytical Chemistry, Chemo- and Biosonsors, University of Regensburg, Germany Robert Zucker, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA The abstracts submitted by the keynote speakers (and participants who have registered early) are published in this issue. (We expect more contributions due to the option of late registration by the end of August which could not be printed in this issue.) I am convinced that their presentations will significantly contribute to a high quality and rewarding scientific program. The complete compilation of abstracts will appear in the local program booklet. Some of the keynote speakers will also offer an additional advanced tutorial. Tutorials are scheduled as follows: Flow Cytometry: Instrumentation, Setup, Adjustment Bead Based Cytometry: Selected Applications Flow Cytometric Analysis of Apoptosis Bacterial Activity Analysis: Proliferation vs. Viability Tests Multiparametric DNA Flow Cytometry of Human Carcinomas Advances in Quantitative Slide Based Cytometry: Towards Cytomics Designing, Implementing, and Analyzing Multicolor Flow Experiments FRET in Flow and Image Cytometry Multiparametric Fluorescence-in-situ-Hybridization (FISH) and Spectral Karyotyping (SKY) in Diagnosis of Human Malignancies Introduction into small animal imaging techniques - a comparative overview It is a great pleasure for me to announce a distinguished lecture that will be given by Stefan Hell, MPI for Biophysical Chemistry, Dept. of NanoBiophotonics (Goettingen, Germany). Stefan Hell is laureate of the “German Annual Technology and Innovation Future Award 2006” which was conferred for the tenth time last year. The award is given in recognition of projects that not only have revolutionary implications for science but are also ready for application and marketing. Stefan Hell was the first to find a way of overcoming the 130-year-old Abbe limit in the fluorescence microscope, the most important microscope in biomedical research. Ever since the 17th century the light microscope has been one of the main symbols of scientific progress – particularly in biology and medicine. Harnessing Stimulated Emission Depletion (STED) microscopy, molecules can now be imaged with far greater definition than ever before. I cordially invite you to attend Stefan Hell's distinguished lecture entitled: “Breaking Abbe's barrier: Diffraction unlimited resolution in far-field microscopy” (probably on Thursday afternoon; please check the final program). It is a long-standing tradition that DGfZ meetings integrate industrial exhibitors. The DGfZ conference could not be held without the participation and sponsorship of industrial companies. Thus, in advance I would like to express my gratitude to the industry for participating in and thereby making our meeting possible. I am sure all will greatly benefit from the industry exhibitions and invite all participants to keep abreast of current state-of-the-art technological developments by visiting the booths and encourage everyone to attend the informative industrial tutorials. No DGfZ meeting is complete without a poster session! It is an essential part of the conference and an excellent opportunity to establish new contacts and interact and discuss important research issues with the presenter of the scientific work in person. Take advantage of this valuable opportunity and visit the poster presentation in a relaxed atmosphere. At the end of the meeting a poster prize will be awarded to an extraordinary presentation both in terms of scientific quality and poster layout. The prestigious Klaus-Goerttler prize was established in 1996 and since then has been awarded for an outstanding Ph.D. thesis or an equivalent work on the occasion of the meeting. I am looking forward to the awarding of this year's prize based on the review and decision by the society's council board. Finally, I would like to express my special gratitude to the local organizing group: Marietta Bock, Simone Diermeier-Daucher, Andrea Sassen, Angelika Graf, Silvia Seegers, Elisabeth Schmidt-Brücken, and Arabel Vollmann-Zwerenz. They have all contributed significantly to the preparation of this year's meeting. Thanks so much. Again, welcome to Regensburg and welcome to the 17th Annual Meeting of the German Society of Cytometry (DGfZ) hosted by the University of Regensburg Medical Center. I hope you will have a fruitful meeting and enjoy the conference as well as the special social program. Cordially yours Gero Brockhoff (President of the German Society of Cytometry) DGfZ ABSTRACTS DISTINGUISHED LECTURE In 1873, Ernst Abbe discovered that the resolution of focusing (‘far-field’) optical microscopy is limited to d = λ/(2 nsin α) > 200 nm, with nsin α denoting the numerical aperture of the lens and λ the wavelength of light. While the diffraction barrier has prompted the invention of electron, scanning probe, and x-ray microscopy, in the life sciences 80% of all microscopy studies are still performed with lens-based (fluorescence) microscopy. The reason is that the 3D-imaging of the interior of (live) cells requires the use of focused visible light. Hence, besides being a fascinating physics endeavor, the development of a far-field light microscope with nanoscale resolution would facilitate observing the molecular processes of life. In this talk, I will discuss novel physical concepts that radically break the diffraction barrier in focusing fluorescence microscopy. They share a common strategy: exploiting selected molecular transitions of the fluorescent marker to neutralize the limiting role of diffraction. More precisely, they establish a certain, signal-giving molecular state within subdiffraction dimensions in the sample [1]. The first viable concept of this kind was Stimulated Emission Depletion (STED) microscopy. In its simplest variant, STED microscopy uses a focused beam for fluorescence excitation, along with a red-shifted doughnut-shaped beam for subsequent quenching of fluorescent molecules by stimulated emission. Placing the doughnut-beam on top of its excitation counterpart in the focal plane confines the fluorescence near its central zero where stimulated emission is absent. The higher the doughnut intensity, the stronger is the confinement. In fact, the spot diameter follows , with I denoting the intensity of the quenching (doughnut) beam and I s giving the value at which fluorescence is reduced to 1/e. Without the doughnut (I = 0) we have Abbe's equation, whereas for I/I s → ∞ it follows that d → 0, meaning that the fluorescence spot can be arbitrarily reduced in size. Translating this subdiffraction spot across the specimen delivers images with a subdiffraction resolution that can, in principle, be molecular! Thus, the resolution of a STED microscope is no longer limited by λ, but on the perfection of its implementation. We will demonstrate a resolution down to λ/45 ≈ 15–20 nm with nanoparticles and biological samples, i.e., 10–12 times below the diffraction barrier. The concept underlying STED microscopy can be expanded by employing other molecular transitions that control or switch fluorescence emission, such as (i) shelving the fluorophore in a metastable triplet state, and (ii) photoswitching (optically bistable) marker molecules between a ‘fluorescence on’ and a ‘fluorescence off’ conformational state. Examples for the latter include photochromic organic compounds, and fluorescent proteins which undergo a photoinduced cis-trans isomerization or cyclization reaction. Due to their optical bistabilty/metastabilty, these molecules entail low values I s, meaning that the diffraction barrier can be broken at low I. A complementary approach is to switch the marker molecules individually and assemble the image molecule by molecule. By providing molecular markers with the appropriate transitions, synthetic organic chemistry and protein biotechnology plays a key role in overcoming the diffraction barrier. Finally, I discuss more recent work of my group showing that the advent of far-field ‘nanoscopy’ has already solved fundamental problems in (neuro)biology, such as the fate of synaptic vesicle proteins after synaptic transmission. Besides, the emerging far-field ‘optical nanoscopy’ also has the potential to advance nanolithography, the colloidal sciences, and to help elucidate the self-assembly of nanosized materials. Reference [1] S.W. Hell, Far-field optical nanoscopy, Science 316 (2007) 1153. INVITED LECTURES Monolayer cell-based assays have become an integral component in many stages of anti-tumor drug testing. However, they still represent a highly artificial cellular environment. 3-D cultures better reflect the pathophysiological in vivo situation in tumor tissues and are increasingly recognized as sophisticated tools for evaluating therapeutic intervention. Multicellular spheroids, one of the classical and well-established 3-D culture systems, reflect some phenomena in tumor tissues that are known to critically affect therapeutic efficacy, such as 3-D cell-cell interactions, development of hypoxic areas and proliferation gradients. Accordingly, they have frequently been applied to anti-tumor therapy testing throughout the past four decades. Nonetheless, their implementation into mainstream drug screening operations is still limited for various reasons. Technological progress with the use and scale-up of the spheroid model in experimental therapeutics include the validation of a reliable tool to rapidly analyze cell viability in tumor spheroids of different sizes. We explored a panel of standard assays to finally provide an easy-handling, standardized protocol that is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for various tumor cell line spheroid types as a basis for a “Spheroid-based Screen”. The intriguing observation that some primary tumor cell populations must be maintained in 3-D culture in order to retain certain tumor initiating (stem) cell properties, adds an additional fascinating challenge for future therapeutic campaigns but of course also requires further evidence and extended studies. 5-Aminolevulinic Acid (ALA) is a precursor of heme in its intracellular biosynthesis. As an intermediate product, the fluorochrome and potent photosensitizer Protoporphyrin IX (PpIX) is produced and can be accumulated under certain conditions. In a number of inflammatory or malignant tissues, this accumulation can be stimulated selectively compared to normal surrounding tissue by topical or systemic delivery of ALA or suitable derivatives. Photodynamic Therapy (PDT) exploits the generation of Singlet-Oxygen by excited PpIX-molecules. For this purpose, intense but non-thermal visible light is irradiated to the sensitized target-tissue. Fluorescence Diagnosis (FD) exploits the low but sufficient fluorescence yield of PpIX to detect and localize otherwise invisible (early) malignant changes. Its great clinical advantage is its applicability during surgery. Preclinical research on PDT and FD has started in 1990. Clinical approval is currently granted in Dermatology for PDT of actinic keratosis and basal cell carcinomas, FD for bladder cancer, and is pending for FD of malignant glioma (fluorescence guided resection). The main steps of preclinical research and current clinical experience will be presented, with special emphasis on developments initiated in Munich. This covers FD and PDT for bladder cancer as well as for malignant glioma, cervical and ovarian cancer and cancer in the oral cavity. Significant clinical experience has already been generated for FD of bladder cancer, proving its superior detection of early high grade cancer. Therapy of bladder cancer guided by intraoperative FD results in a significant reduction of residual tumor rate and an increase of recurrence free survival. The same is apparently true for FD of malignant glioma. A promising current achievement is stereotactic interstitial PDT of recurrent gliomas. A phase I study is showing rather unexpected longterm survival of some of the patients. Further research and development will have to focus on elucidation of PDT-mechanisms (e.g. role of immune response), light delivery instrumentation, consideration of patient individual drug accumulation and optical tissue properties, optimized protocols for PDT and their integration into clinical routine. Two steps led to our present view of the cytoskeleton as a highly dynamic structure that is actively involved in force generation for various kinds of cell motility and is a highly dynamic structure itself. 1. The introduction of video microscopy, especially of the Allen Video Enhanced Contrast-Differential Interference Contrast Microscopy (AVEC-DIC), which allows the visualization of cellular structures in the light microscope that are up to 10 times smaller than the limit of resolution. This enables one to see images of unfixed, unstained, native or purified microtubules and actin bundles, and their interaction with membrane-bounded organelles. Additional information is obtained by applying Dynamic Phase Microscopy in the form of a Linnik type interference microscope and by particle tracking analyses. 2. The discovery of a system exceptionally well-suited to study microtubule and organelle movements, namely, the extruded axoplasm of the squid giant axon. From this axon the cytoplasm can be extruded free from surrounding plasma membrane, and individual microtubules and organelles can be separated from the bulk axoplasm. These techniques which are best suited to study the living cytoplasm are presented together with some of the major results obtained, especially the microtubule-based and the actin filament-based motor enzymes, the dynamic instability of microtubules and a classification of the various types of organelle motility. Emphasis will be laid on the aspect that, except for the Dynamic Phase Microscopy, these techniques which allow us to extend the microscope's ability well beyond the classical limitations of resolution, visualization, brightness and contrast can be retrofitted to regular research microscopes. References Weiss DG, Maile W, Wick RA, Steffen W. Video Microscopy. (Chapter 3). In: Light Microscopy in Biology, A Practical Approach. Lacey AJ (2nd ed.). Oxford University Press, Oxford. 1999; 73–149. Weiss DG, Tychinsky VP, Steffen W, Budde A. Digital light microscopy techniques for the study of living cytoplasm. (Chapter 12). In: Image Analysis: Methods and Applications. Häder DP (ed.). CRC Press, Boca Raton. 2000; 209–239. Weiss DG. Video-enhanced contrast microscopy. (Volume III Chapter 6). In: Cell Biology: A Laboratory Handbook. Celis JE (ed.). Academic Press, 3rd ed. 2005; 57–65. Vaccine development to protect against HIV development is actively proceeding on two fronts: generation of a sterilizing (neutralizing) humoral response, and generation of an effective cellular response. To date, it has been impossible to generate an antibody response of sufficient potency that sterilizing vaccination is possible. Nonetheless, a vaccine can be considered successful on a global scale if the induced cellular response is sufficient to dampen viral loads (reducing transmission) as well as reducing morbidity and mortality after infection. Using different vaccine regimens, we can induce a variety of T cell responses. Using animal models, we hope to identify the kinds of responses that are best correlated with protection against challenge. As we move forward through phase I and II clinical trials in humans, and prepare for phase III, we are keen to determine whether or not these types of responses are induced in humans as well. Our primary tool for determining the quantity and quality of the T cell response is flow cytometry. We routinely measure five or more different functional responses simultaneously from each cell (e.g., cytokine profile, cytotoxic potential, proliferative capacity). These complex combinations of functions reveal that there are a number of distinct “flavors” of T cell responses present in either naturally infected or vaccinated subjects; the task now is to identify which of these flavors is most suited to protection from challenge. In addition, by studying rapid HIV progressors vs. long term nonprogressors, we can identify differences in T cell responses correlated with disease pathogenesis, perhaps pointing us towards desirable types of vaccine responses. These analyses have revealed that a potential correlate of effective T cell responses may be the capacity of T cells to be “polyfunctional”, i.e., to simultaneously effect multiple functions. Polyfunctional T cell responses are correlated with nonprogression in HIV disease, as well as effective viral control of CMV, EBV, and other viruses. In addition, the level of polyfunctional T cells induced by different vaccine regimens directly predicts control of a L. Major infection in mice. The mechanisms accounting for better protection by polyfunctional T cells remain to be elucidated; we are actively characterizing these cells and their differentiation capabilities. In addition, we have identified vaccine regimens that can elicit these cells. Together, these are important tools for the development of novel T-cell based vaccines against pathogens. The ErbB2 (HER2) protein is a member of the EGF receptor (ErbB) family of transmembrane receptor tyrosine kinases. Although no direct ligand has yet been assigned to ErbB2, recent biochemical and biophysical evidence suggests that this protein operates as a shared receptor subunit with other ErbB proteins. Its medical importance stems from its frequent overexpression in breast and other cancers. Humanized antibodies against ErbB2 (i.e. Herceptin or trastuzumab) have been introduced into clinical practice and were found to have cytostatic effect in ˜40% of ErbB2 positive breast tumors. We used tratuzumab resistant (JIMT-1, MKN-7) and sensitive (SKBR-3, N-87) cell lines in order to demonstrate the importance of association pattern ErbB molecules with each other and with integrins, CD44 and lipid rafts. ErbB2, CD44 and beta1-integrin showed significant colocalization with each other and with lipid rafts regardless the cell lines. Trastuzumab-sensitive cell lines expressed more ErbB2 and fewer beta1-integrin and CD44 molecules on their surface than their resistant counterparts. We have found that in the resistant cell lines active ErbB2 homodimers that bind Herceptin with high affinity are scarce. We examined the role of antibody mediated cellular cytotoxicity (ADCC) using JIMT-1 cells that are ErbB2 positive but intrinsically resistant to trastuzumab in vitro. Unexpectedly, trastuzumab was able to inhibit the outgrowth of macroscopically detectable xenograft tumors for up to 5–7 weeks. The effect is likely to be mediated via ADCC, since trastuzumab-F(ab′)2 was ineffective in this model. These results suggest that ADCC may be the predominant mechanism of trastuzumab action on submacroscopic tumor spread. Thus, measuring the ADCC activity of patient's leukocytes against the tumor cells may be a relevant predictor of clinical trastuzumab responsiveness in vivo. Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning approaches. However, the imaging of whole-mount tissues presents technical problems of its own. One of the major problems using CLSM to image whole organs and embryos is tissue penetration of laser light. High quality morphological images begin by optimizing the sample preparation technique [1, 2]. Additional factors include evaluating CLSM performance by optimizing the acquisition variables (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration.) of the confocal microscope [3, 4]. Confocal microscopy has been used by our laboratory to study cell death and morphology in embryos, ovaries, eyes, ears, kidneys lungs and limbs [1, 2]. The technique has revealed structural morphology and the initiation of cell death by the upt
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